P398: DEVELOPMENT AND CHARACTERIZATION OF PRECLINICAL IN VIVO MODELS FOR THE IDENTIFICATION AND TESTING OF NEW THERAPEUTIC APPROACHES FOR PEDIATRIC ACUTE MYELOID LEUKEMIA
Authors: A. Da Ros, V. Indio, E. Porcù, G. Borella, M. Benetton, C. Tregnago, G. Longo, S. Cairo, B. Michielotto, S. Bresolin, B. Buldini, A. Pession, F. Locatelli, M. Pigazzi
Journal: HemaSphere (2022)

Background: In pediatric acute myeloid leukemia (AML) chemotherapy is the standard of care, but >25% of patients still relapse and after a disease recurrence the survival probability is extremely low (<50%). To ameliorate patients’ outcome there is an urgent need to discover new treatments. Nevertheless, pediatric drug development is extremely reduced by the need of a better understanding of the adverse event profile of adult cancer indications in children, by the lack of pediatric-specific formulations, and by the reduced number of pediatric AML patients that can be included in clinical trials. Thus, robust preclinical AML models to faithfully predict new drug efficacy is urgently needed to advance new drugs in clinical setting. Aims: This study aims to generate and characterize AML patient derived xenografts (PDXs) and accelerate the evaluation of innovative medicines for AML. Methods: We generated PDXs from primary AML samples by inoculating blasts in NSG mice and, when engrafted, in 3 consequent mice recipients (namely P0, P1 and P2-PDX). We characterized AML by immunophenotipic profile and by RNA and whole-exome sequencing (WES). According to somatic mutations and copy number alterations we determined AML clonal compositions. We selected drugs and performed drug testing in vivo, after the expansion of P3-PDXs. Results: We generated 22 AML-PDXs representing high-risk AML subtypes for genetic characterization harboring NUP98-NSD1, or NPM1-MLF1, or CBFA2T3-GLIS2, or FUS-ERG or KMT2A somatic translocations, or FLT3-ITD mutation. We monitored the AML associated immunophenotype in PDXs finding it was similar to that of the original AML. By WES we detected a consistent number of variants in each patients’ AML (ranging from 28 to 69), confirming an high AML intra-tumoral heterogeneity. Furthermore, we did not find any mutation recurrence among models, underlining an high inter-tumoral heterogeneity. In all models we tracked clonal evolution from patients’ AML to P2-PDX highlighting that most of the variants were maintained, with very few variants acquired during model development. Monitoring clonal dynamics we recognize a specific “founder” clone characterized by an average of 30 variants which are maintained up to P2 at the same allelic frequency, other small clones with average of 10 variants increasing the allelic frequencies in P2 and, in a restricted number of models, we observed that some clones were lost. By WES and transcriptome analysis we highlighted druggable mutations and pathways allowing the selection of novel targeted drugs. We screened their efficacy in vitro alone or combined with chemotherapic (Arabinoside) and biological agents (Venetoclax) by using AML ex vivo cells and mesenchymal stromal cells in a 3D co-culture system for exploring their synergy in reducing AML proliferation. Four selected drugs are under evaluation in AML-PDX models. Summary/Conclusion: We have created a series of paired AML and xenograft models for advancing pediatric AML therapeutics. Our models represent a concrete perspective for both, the identification of new variants and pathways involved in AML progression, and the possibility to perform novel drug screenings useful to increase AML drug portfolio.